Evaluation of the Murine Local Lymph Node Assay (LLNA) for... the Allergic Contact Dermatitis Hazard Potential of Pesticide Formulations

Evaluation of the Murine Local Lymph Node Assay (LLNA) for... the Allergic Contact Dermatitis Hazard Potential of Pesticide Formulations

Evaluation of the Murine Local Lymph Node Assay (LLNA) for Assessing
the Allergic Contact Dermatitis Hazard Potential of Pesticide Formulations
T McMahon1, D McCall1, J Matheson2, A Jacobs3, T Burns4, D Allen4, W Stokes5
1U.S. EPA, Washington, DC, USA; 2U.S. CPSC, Bethesda, MD, USA; 3U.S. FDA, Silver Spring, MD, USA; 4ILS, Inc., RTP, NC, USA; 5NICEATM/NIEHS/NIH/HHS, RTP, NC, USA
Introduction



Figure 1. LLNA Test Method Protocol
LLNA Peer Review Panel Meetings

The murine local lymph node assay (LLNA) is
a test method for assessing the potential of
substances to cause allergic contact dermatitis
(ACD). ACD is an allergic skin reaction
characterized by redness, swelling, and itching
that can result from repeated contact with a
sensitizing substance.
Public meetings of an international independent
scientific peer review panel (“Panel”) organized by
NICEATM and ICCVAM were held at the Consumer
Product Safety Commission in Bethesda, MD, on
March 4-6, 2008, and at the National Institutes of
Health in Bethesda, MD, on April 28-29, 2009.
Charge to the Peer Review Panel
In its original evaluation of the LLNA, ICCVAM
recommended the LLNA as a valid alternative to
traditionally accepted guinea pig test methods for
assessing ACD hazard potential for most testing
situations (Dean et al. 2001; Haneke
et al. 2001; ICCVAM 1999; Sailstad et al. 2001).
In response to a nomination by the U.S.
Consumer Product Safety Commission in 2007,
NICEATM re-evaluated the applicability domain of
the LLNA.

Review the addendum for errors and omissions

Provide conclusions and recommendations on the
current validation status of the LLNA applicability
domain

Does the information contained in the draft Addendum
support ICCVAM’s draft test method
recommendations?
International Acceptance of Expanded LLNA
Applicability Domain

The LLNA’s expanded applicability domain was included in the updated OECD Test Guideline 429
(OECD 2010) based on ICCVAM’s evaluation.

The revised Test Guideline 429 was adopted by OECD in July 2010 and can be accessed at
http://www.oecd-ilibrary.org/.

Adoption of the revised test guideline is expected to result in broader use of the LLNA, which will
further reduce and refine animal use for ACD assessments while ensuring human safety.
References
Peer Review Panel Conclusions


Table 1. LLNA Performance for Testing
Pesticide Formulations


Comparison
LLNA vs. GP3
n1
23
Accuracy
Sensitivity
LLNA False
Negative Rate
Specificity
LLNA False
Positive Rate
%
No.2
%
No.2
%
No.2
%
No.2
%
No.2
57
13/23
100
3/3
0
0/3
50
10/20
50
10/20

Abbreviations: DPM = disintegrations per minute; SI = stimulation index.
2
3
Considered all of the test materials as candidates for
testing in the LLNA, subject to the limitations outlined
in the ICCVAM draft test method recommendations
Emphasized that before animal testing is conducted,
consideration should be given to the necessity for the
substance to be tested for skin sensitization potential
Recommended including a representative positive
control from the same category of materials to be
tested (e.g., for testing pesticides, select one
representative positive control pesticide)
–
Boverhof D, et al. 2008. Toxicol Sci 105:79-85.
Dean JH, et al. 2001. Regulatory Toxicology and Pharmacology 34(3): 258-273.
Haneke KE, et al. 2001. Regulatory Toxicology and Pharmacology 34(3): 274-286.
ICCVAM 1999. The Murine Local Lymph Node Assay: A Test Method for Assessing the Allergic
Contact Dermatitis Potential of Chemicals/Compounds. Available at:
http://iccvam.niehs.nih.gov/methods/immunotox/llna_PeerPanel98.htm.
ICCVAM. 2009a. Recommended Performance Standards: Murine Local Lymph Node Assay. Available
at: http://iccvam.niehs.nih.gov/methods/immunotox/llna_PerfStds.htm.
ICCVAM. 2009b. The Reduced Murine Local Lymph Node Assay: An Alternative Test Method Using
Fewer Animals to Assess the Allergic Contact Dermatitis Potential of Chemicals and Products.
Available at: http://iccvam.niehs.nih.gov/methods/immunotox/LLNA-LD/TMER.htm.
The complete LLNA Peer Review Panel Reports can
be accessed at:
–
Abbreviations: GP = guinea pig skin sensitization outcomes; No. = number.
1
Concurred that the data supported the ICCVAM draft
test method recommendations for LLNA usefulness
and limitations
http://iccvam.niehs.nih.gov/docs/immunotox_docs/
LLNAPRPRept2008.pdf
http://iccvam.niehs.nih.gov/docs/immunotox_docs/
LLNAPRPRept2009.pdf
OECD. 2010. Guideline For The Testing Of Chemicals - Test Guideline 429: Skin Sensitization: Local
Lymph Node Assay. Available at: http://www.oecd-ilibrary.org/.
Sailstad DM, et al. 2001. Regulatory Toxicology and Pharmacology 34(3): 249-257.
n = Number of substances included in this analysis.
The data on which the percentage calculation is based.
GP refers to outcomes obtained by studies conducted using either the guinea pig maximization test or the Buehler test.
Transmittal to Federal Agencies and Agency
Responses
Acknowledgements
The Intramural Research Program of the National Institute of Environmental Health Sciences
(NIEHS) supported this poster. Technical support was provided by ILS, Inc., under NIEHS contract
N01-ES 35504.

Current Validation Status of the LLNA for
Testing Pesticide Formulations
The data supporting the ICCVAM recommendations is contained in an Addendum to the 1999
evaluation (Appendix C of the ICCVAM Test Method Evaluation Report [ICCVAM 2010]).

In June 2010, ICCVAM forwarded final test method recommendations on the expanded uses of
the LLNA for pesticide formulations and other products to U.S. Federal agencies for
consideration.
This poster reflects the views of the authors. The views expressed above have not been reviewed or
approved by the U.S. Consumer Product Safety Commission or any other U.S. Federal agency and
do not necessarily represent the official positions of any U.S. Federal agency.


Federal agency responses include acceptance decisions and agreement with the test method
recommendations for the expanded uses of the LLNA.
Since the poster was written as part of the official duties of the authors, it can be freely copied.

Several agencies also indicated that they would communicate the ICCVAM recommendations to
stakeholders and encourage their appropriate use. For example, EPA has issued a policy on the
use of LLNA for pesticide formulations (see http://www.epa.gov/pesticides/science/llnapolicyfinal.pdf).
NICEATM LLNA database of over 600 substances included data for 104 pesticide formulations.
–
–

Included both LLNA and guinea pig (GP) data on 23 formulations
Did not include human skin sensitization test data or postmarketing sensitization report data
For the 23 formulations with both GP and LLNA data:
–
LLNA and the GP results agreed (accuracy) 57% (13/23) of the time (Table 1).
–
LLNA classified 57% (13/23) of formulations as sensitizers while GP tests classified only 13%
(3/23) as sensitizers.

Independent Scientific Peer Review Panel
NICEATM and ICCVAM gratefully acknowledge the following individuals and institutions that
submitted data to NICEATM to evaluate the LLNA applicability domain.
Agency responses are available on the NICEATM–ICCVAM Web site at
http://iccvam.niehs.nih.gov/methods/immunotox/llna.htm.
 All 3 GP sensitizers were also LLNA sensitizers (i.e., no pesticide formulations were
underpredicted by the LLNA compared to GP results).
–
The LLNA identified 10 formulations as sensitizers that were classified as nonsensitizers in
GP tests (Table 1).
ICCVAM Interagency Immunotoxicity
Working Group
Consumer Product Safety Commission
Joanna Matheson, PhD (Working Group
Co-chair)
Marilyn Wind, PhD (to July 2010)
Test Method Usefulness and Limitations for
Pesticide Formulations

ICCVAM concludes that these data support the usefulness of the LLNA for testing pesticide
formulations.
–
–
–
For adequate dermal exposure during the testing of aqueous formulations, an appropriate
vehicle should be used to facilitate adherence of the test material to the skin (e.g.,
1% Pluronic L92 [Boverhoff et al. 2008]).
If an LLNA variant (e.g., a nonradioactive LLNA version) is validated for use to test novel
substance classes, then the findings should be relevant to the family of validated and
accepted LLNA tests.
As indicated in Table 1, there is a greater likelihood of obtaining a positive result in the LLNA
than in a GP test. Therefore, the potential for possible overclassification may be a limitation
of the LLNA.
Environmental Protection Agency
Office of Pesticide Programs
Jonathan Chen, PhD
John R. “Jack” Fowle III, PhD, DABT
Masih Hashim, DVM, PhD
Marianne Lewis
Deborah McCall
Timothy McMahon, PhD
John Redden
Jenny Tao, PhD
Office of Pollution Prevention and Toxics
Elizabeth Margosches, PhD
Ronald Ward, PhD
Office of Research and Development
Marsha Ward, PhD
Food and Drug Administration
Center for Devices and Radiological Health
Vasant G. Malshet, PhD, DABT
Jeffrey Toy, PhD
Center for Drug Evaluation and Research
Ruth Barratt, PhD, DVM
Paul Brown, PhD
Abigail Jacobs, PhD (Working Group Co-chair)
Jiaqin Yao, PhD
Center for Food Safety and Applied Nutrition
Donnie Lowther
Neil Wilcox, DVM, MPH (to April 2011)
Office of the Commissioner
Suzanne Fitzpatrick, PhD, DABT
National Institute of Environmental Health
Sciences
Warren Casey, PhD, DABT
Dori Germolec, PhD
William Stokes, DVM, DACLAM
National Institute for Occupational Safety
and Health
B. Jean Meade, DVM, PhD
Paul D. Siegel, PhD
Test Method Protocol for Pesticide Formulations


National Library of Medicine
Pertti Hakkinen, PhD
The updated ICCVAM-recommended LLNA test method protocol (Figure 1; Appendix A, ICCVAM
2009a) reduces animal use by 20% compared to the 1999 ICCVAM-recommended protocol
(ICCVAM 1999).
European Centre for the Validation of Alternative
Methods - Liaison
Silvia Casati, PhD
Alexandre Angers, PhD
If dose-response information is not required or there is no basis to believe that the test article
may be a sensitizer, a reduced LLNA test method protocol should be considered. By testing only
the high dose, the reduced LLNA can further reduce animal use by up to 40% (ICCVAM 2009b).
Japanese Center for the Validation of Alternative
Methods - Liaison
Hajime Kojima, PhD
Michael Luster, PhD (Panel Chair)
Senior Consultant to the National Institute
for Occupational Safety and Health
Morgantown, WV
Nathalie Alépée, PhD
L’Oréal Research and Development
Aulnay sous Bois, France
Anne Marie Api, PhD
Research Institute for Fragrance Materials
Woodcliff Lake, NJ
Nancy Flournoy, MS, PhD
University of Missouri–Columbia
Columbia, MO
Thomas Gebel, PhD
Federal Institute for Occupational
Safety and Health
Dortmund, Germany
Sidney Green, PhD
Howard University
Washington, DC
Kim Headrick, BAdmin, BSc
Health Canada
Ottawa, Ontario, Canada
Dagmar Jírová, MD, PhD
National Institute of Public Health
Prague, Czech Republic
David Lovell, PhD
University of Surrey
Guildford, Surrey, U.K.
Howard Maibach, MD
University of California–San Francisco
San Francisco, CA
James McDougal, PhD
Wright State University
Dayton, OH
Michael Olson, PhD
GlaxoSmithKline
Research Triangle Park, NC
Raymond Pieters, PhD
Utrecht University
Utrecht, The Netherlands
Jean Regal, PhD
University of Minnesota Medical School
Duluth, MN
Jonathan Richmond, MB ChB, FRCSEd
Home Office
London, U.K.
Peter Theran, VMD
Consultant, Massachusetts Society for the
Prevention of Cruelty to Animals
Novato, CA
Stephen Ullrich, PhD
M.D. Anderson Cancer Center
Houston, TX
Michael Woolhiser, PhD
Dow Chemical
Midland, MI
Takahiko Yoshida, MD, PhD
Asahikawa Medical College
Hokkaido, Japan
Ann Marie Api, PhD
Research Institute for Fragrance Materials
Woodcliff Lake, NJ
Michael J. Olson, PhD
GlaxoSmithKline
Research Triangle Park, NC
Phil Botham, PhD
European Crop Protection Association
Brussels, Belgium
Kirill Skirda, PhD
TNO Quality of Life
Delft, Netherlands
Eric Debruyne, PhD
Bayer CropScience SA, Sophia Antipolis
Cedex, France
Peter Ungeheuer, PhD
European Federation for Cosmetic Ingredients
Frankfurt, Germany
G. Frank Gerberick, PhD
Procter and Gamble Company
Cincinnati, OH
Michael Woolhiser, PhD
Dow AgroSciences
Midland, MI
Dori Germolec, PhD
National Toxicology Program
Research Triangle Park, NC